This research program has focused on characterizing the synapsin family of phosphoproteins and its regulation of neurotransmitter release. By a variety of in vitro and in vivo techniques, we have demonstrated that synapsins are tightly associated with the cytoplasmic face of synaptic vesicles and can bind simultaneously to both synaptic vesicles and actin. Phosphorylation of synapsin I by calcium/calmodulin-dependent protein kinase II (CaMKII) or MAP kinase decreases its affinity for synaptic vesicles and actin, thus allowing vesicles to move within the vesicle cluster from a reserve pool to a readily releasable pool, where they can participate in exocytosis. In addition, we have recently discovered that synapsins directly modulate the dynamics of the readily releasable pool and thus the kinetics of exocytosis. This revised application for renewal proposes to extend our studies on the mechanisms by which synapsins regulate synaptic transmission. To understand the function(s) of synapsins at the molecular level, we propose the following three Specific Aims: I. To determine the impact of synapsin mutations on neurotransmitter release, synaptic vesicle trafficking and the trafficking to and within nerve terminals. We will make use of GFP-labeled synapsins, site-directed mutagenesis, transient cell transfection techniques and live cell imaging combined with physiological approaches for these purposes. II. To study the regulation of Src-family tyrosine kinases by synapsins in nerve terminals. We have previously demonstrated that synapsins are excellent ligands for the Src homology-3 (SH3) domain of c-Src. and that this interaction leads to activation of endogenous vesicle-associated tyrosine kinase, resulting in phosphorylation of endogenous vesicle substrates including synapsins. III. To identify novel binding partners for specific regions of the synapsins and to study their functional significance. Synapsins have been shown to interact with the SH3 domains of c-Src and Grb2. Preliminary results indicate that synapsin-SH3 domain interactions also include many proteins involved in signal transduction and regulation of membrane trafficking at the nerve terminal. We will use in vitro methods to investigate the interactions of synapsins mediated by SH3 domains of proteins relevant to nerve terminal function. We will also investigate those interactions mediated by the highly conserved domains A, C, and E of synapsins. The effects of acute disruption of the identified synapsin interactions on presynaptic morphology and neurotransmitter release will be determined in the in vivo models of the lamprey reticulospinal synapse and the squid giant synapse.